Research Program A. Background and Preliminary Data: The goal of the research program is to understand the contribution of cell cycle pathways that predicate and dictate the acquisition of malignant characteristics by normal cells. Cyclin-dependent kinase 4 (Cdk4) is a crucial regulator of G1/S cell cycle progression. The importance of the Cdk4 locus in human cancer was emphasized upon identification of germline mutations in CDK4, including the CDK4-Arg24Cys (R24C) mutation, which abolishes the ability of CDK4 to bind to p16INK4a, resulting in melanoma. This observation suggested that a mutant CDK4 gene, that would de-regulate downstream E2F transcription factor pathways, could function as a dominant oncogene that is resistant to normal physiological inhibition. To analyze the role of Cdk4 in normal mouse development and the role of mutant Cdk4 in oncogenesis we have generated two strains of mice carrying a targeted Cdk4 locus. One of the strains does not express Cdk4 (Cdk4neo). Mice from the second strain express normal levels of a p16Ink4a-insensitive mutant Cdk4 protein that carries the R24C mutation (Cdk4R24C) identified in human melanoma patients. Cdk4neo/neo fibroblasts exhibit a delay in reaching the S-phase, indicating that Cdk4 is required for proper entry of resting cells into the cell cycle. Cdk4 knock-out mice suffer early mortality due to defects in growth, fertility and diabetes due to developmental defects in insulin-producing pancreatic beta cells. In contrast, fibroblasts derived from Cdk4R24C/R24C mice that inherit the activated Cdk4R24C mutation have a shorter cell cycle, high proliferation indices, fail to undergo senescence, are capable of long-term growth in culture, can be readily transformed by activated Ras, Myc or E1A oncogenes and form tumors in nude mice. Cdk4R24C/R24C mice exhibit a dramatic and preferential hyperplasia of the endocrine beta-cell islet compartment of the pancreas that progresses to pancreatic adenomas and carcinomas illustrating the critical and highly specific role that Cdk4 activity plays in pancreatic beta cell development and proliferation. By 8-10 months both heterozygous Cdk4+/R24C mice and homozygous Cdk4R24C/R24C mice that harbor the activated Cdk4R24C allele develop tumors of varying etiology, clearly demonstrating the consequence of de-regulated Cdk4 mediated cell cycle pathways in progression of various types of cancers. Among the tumor pathology, we observed a predominance of mammary, pituitary, brain, pancreatic, liver and skin tumors. Taken together, the Cdk4 knock-out' and Cdk4R24C knock-in' mice provide important tools to investigate the contribution of de-regulated G1-S phase cell cycle pathways during carcinogenic progression as well as to provide investigative tools for potential cell cycle directed cancer therapy. B. Area of Investigation # 1. Characterize the alterations in Cell Cycle Pathways during progression from Hyperplasia to Adenoma to Carcinoma. Our published and preliminary observations indicate that de-regulated Cdk4R24C mutation dictates the cellular expansion that drives the cells from Normal to Hyperplasia to Adenoma to Carcinoma. In contrast we have observed that the same cell types are adversely affected and undergo atrophy in the Cdk4 knock-out' mice. Taken together, these observations underlie the causal' effects of Cdk4 in determining the cellular fate destined for either Atrophy or Carcinoma. We plan to identify the critical regulators that lead to these opposing cellular phenotypes. 1. Pancreatic Development and Carcinogenesis (Collaboration with Dr. Franz Matschinsky. University of Pennsylvania. Philadelphia. PA and Dr. Jeff Green, LCRC, NCI). Studies are underway to determine the molecular mechanism that drives the Beta cell progression towards atrophy (Cdk4 knock-out) or carcinoma (Cdk4R24C Knock-in). To this end, we are characterizing the proliferation and apoptosis pathways in beta cells.